Amino acids have been shown to improve the development of pre-implantation embryos in vitro in a variety of species, such as the mouse (Gardner and Lane 1993), rat (Zhang and Armstrong 1990; Miyoshi et al. 1995), sheep (Gardner et al. 1994) and cow (Takahashi and First 1992; Rosenkrans and First 1994; Keskintepe et al. 1995); Lane and Gardner (1994) reported that Eagle's essential amino acids increased the inner cell mass (ICM) cell numbers in murine embryos cultured from the zygote stage.
Embryos in vivo, derive exogenous amino acids from oviducal and uterine fluids. A total of 20 free amino acids have been detected in bovine oviducal fluid (Stanke et al. 1974), and 25 have been detected in bovine uterine fluid (Fahning et al. 1967). Moore and Bondioli (1993) found glycine and alanine to be the two most predominant amino acids in bovine oviducal fluid and that supplementation with these amino acids enhanced bovine embryo development in the presence of oviducal cells. Suh et al. (1995) reported that significantly more bovine zygotes cultured in CR2 medium with glycine reached the blastocyst stage. Rieger and Loskutoff (1994) have shown that glutamine and glycine are consumed by denuded bovine oocytes, and that glutamine is taken up during early pre-implantation development (Rieger et al. 1992).
Although studies in this area have concentrated on administering single or pairs of radiolabelled amino acids, embryos within the female tract will be exposed to a mixture of amino acids (Leese 1988). Lamb and Leese (1994) measured the consumption of a physiological mixture of 20 amino acids by murine blastocysts, and found that 9 were depleted significantly.
The fate of amino acids in bovine embryos has been investigated by Frei et al. (1989) who measured the rate of incorporation of radiolabelled methionine into protein. They found a quantitative decrease in the rate of protein synthesis between the zygote and 8-cell stage, followed by a progressive increase from this point to the blastocyst stage. The quantitative increase in amino acid utilisation observed around these stages of development could be related to the initiation of transcription of the bovine embryonic genome which occurs at the 8-16-cell stage of development (Telford et al. 1990).
Amino acids have also been shown to improve the development of bovine zygotes fertilised in vitro to morulae and blastocysts and to increase total cell numbers at the blastocyst stage (Takahashi and First 1992; Rosenkrans and First 1994; Keskintepe et al. 1995). It is not clear how exogenously-administered amino acids assist embryo development in vitro; some, such as glutamine, may act as energy sources (Rieger and Guay 1988; Rieger 1992), others may increase the pool size of endogenous amino acids and thereby stimulate de novo protein synthesis (Zhang and Armstrong 1990). Van Winkle and Dickinson (1995) have shown that there are significant differences between the amino acid content of murine embryos that develop in vitro and those that develop in vivo.
Partridge and Leese (1996) investigated bovine embryos which had been cultured with 19 amino acids at concentrations routinely used to supplement the medium synthetic oviduct fluid (SOF; Tervit et al. 1972). Groups of embryos fertilised in vitro from the putative zygote stage to the blastocyst stage, and blastocysts freshly flushed from the uterus on Day 7 after fertilisation (derived in vivo) were studied. Depletion rates for 17 of the amino acids were measured over a 12-h period with individual amino acids detected by high performance liquid chromatography (HPLC) following fluorimetric derivatisation.
Partridge and Leese (1996) found glutamine depletion at the putative zygote stage (0.76±0.05 pmol zygote−1 h−1) and at the 4-cell stage (0.94±0.1 pmol embryo−1 h−1). However, a greater depletion of glutamine was not observed at the blastocyst stage, in contrast to the results of Rieger et al. (1992) who measured the uptake of radiolabelled glutamine given as a single amino acid substrate in B2 medium by bovine embryos.
With regard to amino acid depletion, a most intriguing result of Partridge and Leese (1996) was the depletion of threonine in significant amounts at all stages of development in vitro as well as by the blastocyst derived in vitro. The fate of threonine is unknown, but it could act as an energy substrate, by entering the Krebs cycle as acetyl-Coenzyme A (CoA) or succinyl-CoA.
Alanine was produced in significant amounts by all stages of embryos produced in vitro and by embryos derived in vivo. Van Winkle and Dickinson (1995) hypothesised that alanine could act as a route for embryos to sequester waste nitrogen since very high concentrations were found in murine blastocysts grown in vitro. In addition, Gardner and Lane (1993) have shown that ammonia toxicity is a potential problem for mouse embryos grown in vitro. The large increase in external alanine concentration observed during the culture of bovine embryos produced in vitro and derived in vivo in the present study leads us to suggest that alanine may indeed be formed by the embryo to prevent the build-up of toxic ammonium ions.
The inclusion of amino acids in human pre-implantation culture medium has become more prevalent since the advent of blastocyst transfer and the requirement for increased embryo development beyond the 4- to 8-cell stage.
In spite of this, there is still little knowledge regarding which amino acids are actually utilised by the embryo at various stages of development.
Current methods for in-vitro embryo production include in-vitro fertilisation and intra cytoplasmic sperm injection (ICSI). Embryo production may also follow the techniques of cryopreservation and embryo biopsy.
Understanding the way in which embryos modify an amino acid mixture may provide a clue to understanding why the embryo produced in vitro is less robust than its in vivo counterpart. These problems are particularly apparent in human in vitro fertilisation (IVF) programmes whereby the average rate of success in the UK is currently about 17% or 1 in 6.
A typical human IVF programme involves the administration of ovarian egg production and releasing hormones to the woman. These eggs are collected and inseminated with sperm to generate about ten embryos. Up to three (in the UK) of the fertilised embryos will then be transferred back to the woman and if the programme is successful, at least one will implant itself in the womb and continue to develop.
In an effort to reduce hormone administration, eggs may be collected at the earlier stages of oogenesis. Subsequent maturation of the eggs occurs in-vitro. Following insemination of the in-vitro matured eggs, up to three fertilised embryos are then transferred back to the woman for implantation.
The method of intra cytoplasmic sperm injection is now increasingly used for fertilisation. Subsequent to the administration of ovarian egg production and releasing hormones, eggs, surrounded by cumulus cells, are released. The protective layer obscures the egg and must be removed to reveal an egg which is then subjected to a system of visual grading before sperm injection is carried out.
To date there exists no method by which embryos or eggs with increased development potential can be effectively and reliably selected, although glucose consumption and lactate production have been used in the mouse for this purpose. Comparative studies of physiological parameters such as glucose, pyravate, or oxygen consumption in arresting and healthy embryos or eggs have failed to provide a solution to the problem. Current methods rely on morphological selection whereby embryos and eggs are subjected to a grading system.
Because of the great uncertainty in the determination of the most viable embyros and eggs, the need to transfer more than one embryo back into the mother for implantation after artificial insemination becomes apparent. This procedure compensates for the likelihood that one or more of the embryos may fail to develop and serves to heighten the limited chances of success.
Increasing the reliability of the egg or embryo selection will have important ramifications upon the IVF programme as a whole whereby the most viable embryo can be selected and transferred for subsequent implantation. The transfer of a single viable embryo guards against the possibility of multiple births which carries the risk of premature birth and perinatal problems.
It should be understood that any test does not need to be 100% accurate or reliable but should simply provide a non invasive method for consistent indication as to the viability of a single egg or embryo.
A suitable test should involve a selection period which is as short as possible so that transfer of the embryo and implantation can take place as soon as possible after in vitro fertilisation. This minimises any risks which might be associated with prolonged exposure of the developing embryo to the artificial culture conditions. A shorter selection period is also beneficial from an economic point of view because the costs of an otherwise labour and resource intensive operation can be minimised.
Considerable research interest is also focused on the generation of embryos by nuclear transfer (NT). Such embryos are made by injecting a nucleus from a donor cell (karyoplast) into an enucleated egg (ooplast) and then using an electric pulse to trigger embryo development. A variety of karyoplasts have been used for nuclear transfer including stem cells, which are derived from the inner cell mass of the blastocyst and which are the precursor cells for all tissues of the body. However, embryo-derived stem cells (ES cells) have only been conclusively isolated from the mouse and the human and there is an intensive search for methods to produce them in other species including the domestic species. In the case of ‘Dolly’ the karyoplast was a somatic (adult) mammary gland cell.
The generation of embryos by nuclear transfer, especially from stem cells, is the preferred route towards the production of transgenic animals and for cell ‘therapeutic cloning’—the production of new cells and tissues to replace those which have become diseased or ceased to function properly. However, current methods for the production and identification of karyoplasts, stem cells, stem cell precursors and viable nuclear transfer embryos are laborious and time consuming.
There is a need for a biochemical marker(s) which would simplify the identification of a cell such as a gamete (which may be at any stage of development), an embryo (which may be made by nuclear transfer), a karyoplast, a putative stem cell population, a stem cell precursor population or a stem cell population.
As used herein the term ‘egg’ refers to an egg at any stage of oogenesis and includes in-vitro matured eggs.